Influenza is a worldwide public health problem. Although current influenza vaccines are effective in battling closely matched viruses, major limitations of current vaccines are the need to produce new vaccines every season, the uncertainty in choice of the correct strains, and the inability to prevent a new influenza pandemic. Improved vaccines inducing broadly protective immune responses against multiple type A influenza viruses are urgently needed, not only for seasonal influenza but also for pandemic influenza prevention. Conserved epitopes are potent immunogens for such vaccines. Combined strategies including improved antigens, new antigen delivery techniques and vaccination regimens should be considered together to overcome current obstacles in the development of a universal influenza vaccine. In the project we propose to generate constructs encoding conserved influenza antigens which are not usually sensed the by host immune system in natural viral infection and seasonal vaccination. We will use the newest nanotechnology approach to produce nanoclusters self-assembled directly from the resulting antigenic proteins as universal influenza vaccine candidates. We will investigate the breadth of cross-protection induced by these nanovaccines in mouse and guinea pig models, and the immune correlate of the cross protection. The three specific aims to be investigated are: 1. Generation of constructs expressing conformation-stabilized HA stalk domain (csHA-stalk) and tetrameric M2e tandem repeat (tM2et) recombinant proteins. The relatively conserved stalk domain in HA is shielded by its globular head domain in virions and is less effectively sensed by immune cells. A csHA-stalk without the globular head will overcome this limitation, and retain its native conformation. Also, tM2et will retain the tetrameric structure of M2e with increased epitope density. 2. Production of nanoclusters self-assembled from csHA-stalk and tM2e antigens. These nano-size particles will be directly self-assembled from the resulting antigenic recombinant proteins, and release intact antigens after uptake. These particles will maximize antigenic payload, control spatial antigen presentation, and regulate release in DCs. We will characterize the size of the particles, antigen content, distribution and release, and internalization by DCs. 3. Investigation of immune responses and the breadth of protective immunity induced by the above nanoclusters and of immune correlates of broad cross-protection. Vaccines based on a combination of HA stalk domains from the two phylogenetic groups may protect against all influenza A viruses. The addition of tM2et will further increase the potential for broad protection. We will study immune responses induced by different nanovaccine combinations, the cross protection to challenge by a panel of influenza A viral strains, and immune correlates of the cross protection.